Glycerol is a promising low-cost solvent for biomass pretreatment since a large amount of glycerol is generated as a by-product in the biodiesel industry. Pretreatment is a method of disintegration of the recalcitrant structure of biomass to enhance the accessibility of cellulose and hemicelluloses to enzymes for complete saccharification. During pretreatment, glycerol breaks the lignin carbohydrate complex and selectively solubilizes lignin. Thus, the glycerol pretreatment improves the accessibility of cellulose to cellulases leading to higher sugar yields. The glycerol pretreatment is carried out at high temperature (>190 °C) to disintegrate the structure of biomass. The glycerol pretreatment in the presence of acid or base catalyst such as H2SO4 or NaOH results in lower pretreatment temperature and higher glucan hydrolysis. This chapter describes the methodology to carry out glycerol pretreatment of sorghum biomass with or without acid/alkali as catalyst and the basic calculations to determine the efficiency of the pretreatment. © 2021, Springer Science+Business Media, LLC, part of Springer Nature.